WO2015060314A1 - Method for producing l-amino acid - Google Patents

Abstract

Provided is a method for producing an L-amino acid. An L-amino acid can be produced by culturing a bacterium that belongs to the family Enterobacteriaceae, is so modified as to weaken an acpP-fabF operon and is capable of producing an L-amino acid in a culture medium and then collecting the L-amino acid from the culture medium or cells of the bacterium.

Description

Method for producing L-amino acid

The present invention relates to a method for producing L-amino acids using bacteria. L-amino acids are industrially useful as additives for animal feed, ingredients for seasonings and foods and drinks, amino acid infusions, and the like.

L-amino acids are industrially produced, for example, by fermentation using various microorganisms capable of producing L-amino acids. Examples of methods for producing L-amino acids by fermentation include a method using a wild-type microorganism (wild strain), a method using an auxotrophic strain derived from a wild strain, and various drug-resistant mutant strains derived from a wild strain. And a method using a strain having characteristics of both an auxotrophic strain and a metabolic control mutant.

In recent years, microorganisms whose L-amino acid producing ability has been improved by recombinant DNA technology have been used for the production of L-amino acids. Examples of a method for improving the L-amino acid producing ability of a microorganism include, for example, enhancing the expression of a gene encoding an L-amino acid biosynthetic enzyme (Patent Documents 1 and 2) or an L-amino acid biosynthetic system. To enhance the inflow of the carbon source (Patent Document 3).

The acpP gene is a gene encoding an acyl carrier protein (ACP) (Non-patent Document 1). ACP is translated as an inactive apo-ACP, and then converted into a 4′-phosphopantethein (4′−) at the serine residue at position 36 (in the case of Escherichia coli) of apo-ACP by ACP synthase (ACP synthease). phosphopanteheine) is added as a cofactor to form active holo-ACP. ACP is a protein that plays an important role in fatty acid biosynthesis of bacteria and the like. Specifically, ACP (holo-ACP) binds to a fatty acid chain via a 4'-phosphopantethein group and carries the fatty acid chain during fatty acid biosynthesis.

The fabF gene is a gene encoding β-ketoacyl-ACP synthase II (non-patent document 1). β-ketoacyl-ACP synthase II is one of fatty acid biosynthetic enzymes and is involved in the elongation of fatty acid chains. Specifically, β-ketoacyl-ACP synthase II catalyzes a reaction for producing 3-oxoacyl-ACP (carbon number n + 2) from acyl-ACP (carbon number n) and malonyl-ACP (EC 2.3.1.41). ).

In Escherichia coli, a group of genes involved in fatty acid biosynthesis including the acpP gene and the fabF gene exists as a yceD-rpmF-plsX-fabHDG-acpP-fabF gene cluster. The gene group of the cluster is co-transcribed as several gene pairs (Non-patent Document 1). For example, the acpP gene and the fabF gene are cotranscribed as the acpP-fabF operon. The fabF gene is also transcribed individually from its own promoter. The acpP gene can also be co-transcribed from the fabD gene and the fabG gene in the yceD-rpmF-plsX-fabHDG-acpP-fabF gene cluster.

However, the relationship between the acpP gene and fabF gene and L-amino acid production has not been known.

U.S. Pat.No. 5,168,056 U.S. Pat.No. 5,776,736 U.S. Pat.No. 5,906,925

An object of the present invention is to develop a novel technique for improving L-amino acid-producing ability of bacteria and to provide an efficient method for producing L-amino acid.

As a result of intensive studies to solve the above problems, the present inventor can improve the L-amino acid-producing ability of bacteria by modifying the bacteria so that the expression of acpP and fabF genes is reduced. The present invention was completed.

That is, the present invention can be exemplified as follows.
[1]
Bacteria belonging to the family Enterobacteriaceae having L-amino acid-producing ability are cultured in a medium to produce and accumulate L-amino acid in the medium or in the bacterial body, and L-amino acid is produced from the medium or the bacterial body. Collecting an L-amino acid comprising:
A method, characterized in that the bacterium has been modified such that the acpP-fabF operon is attenuated.
[2]
The method, wherein the weakening of the acpP-fabF operon is a decrease in the activity of a protein encoded by a gene of the acpP-fabF operon.
[3]
The method, wherein the expression of the acpP-fabF operon gene is weakened to weaken the operon.
[4]
The method, wherein expression of the gene is weakened by modifying an expression control sequence of the gene of the acpP-fabF operon.
[5]
The method, wherein the gene for the acpP-fabF operon is an acpP gene and / or a fabF gene.
[6]
The method, wherein the genes for the acpP-fabF operon are an acpP gene and a fabF gene.
[7]
The method, wherein the expression of the acpP-fabF operon gene is weakened by replacing cytosine at position −34 upstream of the translation start point of the acpP gene with another base.
[8]
The method, wherein the expression of the acpP-fabF operon gene is attenuated by replacing cytosine at position −34 upstream of the translation start point of the acpP gene with adenine.
[9]
The method, wherein the bacterium belongs to the genus Escherichia, Pantoea, or Enterobacter.
[10]
The method, wherein the bacterium is Escherichia coli.
[11]
The method, wherein the L-amino acid is L-lysine.
[12]
Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or cells of the Escherichia coli, and L-lysine is collected from the medium or cells. A process for producing L-lysine comprising
A method wherein the expression of the gene is weakened by modifying the expression control sequence of the gene of the acpP-fabF operon in the Escherichia coli.
[13]
Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or cells of the Escherichia coli, and L-lysine is collected from the medium or cells. A process for producing L-lysine comprising
A method wherein cytosine at position −34 upstream of the translation start point of the acpP gene is substituted with another base in the Escherichia coli.
[14]
Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or cells of the Escherichia coli, and L-lysine is collected from the medium or cells. A process for producing L-lysine comprising
A method wherein cytosine at position −34 upstream of the translation start point of the acpP gene is substituted with adenine in the Escherichia coli.

Hereinafter, the present invention will be described in detail.

The method of the present invention comprises culturing a bacterium belonging to the family Enterobacteriaceae having L-amino acid-producing ability in a medium, and producing and accumulating L-amino acid in the medium or in the microbial cells, and the medium or the bacterium. A method for producing an L-amino acid comprising collecting an L-amino acid from a body, wherein the bacterium is modified so that the acpP-fabF operon is weakened. The bacterium used in this method is also referred to as “the bacterium of the present invention”.

<1> Bacteria of the Present Invention The bacterium of the present invention is a bacterium belonging to the family Enterobacteriaceae having an L-amino acid-producing ability and modified so that the acpP-fabF operon is weakened.

<1-1> Bacteria having L-amino acid-producing ability In the present invention, “bacteria having L-amino acid-producing ability” refers to the extent that a desired L-amino acid can be produced and recovered when cultured in a medium. Refers to bacteria having the ability to accumulate in the medium or in the fungus body. The bacterium having L-amino acid-producing ability may be a bacterium capable of accumulating a larger amount of the target L-amino acid in the medium than the unmodified strain. Non-modified strains include wild strains and parent strains. The bacterium having L-amino acid-producing ability is a bacterium that can accumulate the target L-amino acid in an amount of 0.5 g / L or more, more preferably 1.0 g / L or more in the medium. May be.

L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, glycine, etc. Aliphatic amino acids, amino acids which are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan, L- Examples thereof include sulfur-containing amino acids such as cysteine, L-cystine and L-methionine, acidic amino acids such as L-glutamic acid and L-aspartic acid, and amino acids having an amide group in the side chain such as L-glutamine and L-asparagine. The bacterium of the present invention may have only one L-amino acid producing ability or may have two or more L-amino acid producing ability.

In the present invention, the term “amino acid” may mean an L-amino acid unless otherwise specified. Further, the produced L-amino acid may be a free form, a salt thereof, or a mixture thereof. That is, in the present invention, the term “L-amino acid” may mean a free L-amino acid, a salt thereof, or a mixture thereof, unless otherwise specified. Examples of the salt will be described later.

The bacteria belonging to the family Enterobacteriaceae include Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photolabdas Examples include bacteria belonging to genera such as (Photorhabdus), Providencia, Salmonella, Morganella, and the like. Specifically, according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) Bacteria classified in the family Enterobacteriaceae can be used.

The Escherichia bacterium is not particularly limited, but includes bacteria classified into the genus Escherichia by classification known to microbiologists. Examples of Escherichia bacteria include, for example, Neidhardt et al. (Backmann, B. J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Nehard (ed.), “Escherichia, coli, and Salmonella, Cellular, and Molecular, Biology / Second Edition, American, Society, for Microbiology, Press, Washington, DC). Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Specific examples of Escherichia coli include, for example, Escherichia coli W3110 (ATCC エ 27325) and Escherichia coli MG1655 (ATCC プ ロ ト タ イ プ 47076) derived from the prototype wild type K-12.

The bacteria belonging to the genus Enterobacter are not particularly limited, but include bacteria classified into the genus Enterobacter by classification known to microbiologists. Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans include the Enterobacter agglomerans ATCC12287 strain. Specific examples of Enterobacter aerogenes include Enterobacter aerogenes ATCC13048, NBRC12010 (BiotechonolonBioeng.eng2007 Mar 27; 98 (2) 340-348), AJ110637 (FERM BP-10955) . Examples of Enterobacter bacteria include those described in European Patent Application Publication No. EP0952221. Some Enterobacter agglomerans are classified as Pantoea agglomerans.

The Pantoea bacterium is not particularly limited, and examples include bacteria classified into the Pantoea genus by classification known to microbiologists. Examples of the genus Pantoea include Pantoea 、 ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specifically, for example, Pantoea Ananatis LMG20103 strain, AJ13355 strain (FERM 、 BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), SC17 strain (FERM BP) -11091), and SC17 (0) strain (VKPM B-9246). Certain types of Enterobacter agglomerans were recently reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc. based on 16S rRNA nucleotide sequence analysis (Int. J. Syst. Bacteriol) ., 43, 162-173 (1993)). In the present invention, the Pantoea bacterium also includes a bacterium reclassified as Pantoea in this way.

Examples of the genus Erwinia include Erwinia amylovora and Erwinia carotovora. Examples of Klebsiella bacteria include Klebsiella planticola.

These strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

The bacterium of the present invention may inherently have L-amino acid-producing ability or may have been modified to have L-amino acid-producing ability. A bacterium having L-amino acid-producing ability can be obtained, for example, by imparting L-amino acid-producing ability to the bacterium as described above, or by enhancing the L-amino acid-producing ability of the bacterium as described above. .

L-amino acid-producing ability can be imparted or enhanced by a method conventionally used for breeding amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Academic Publishing Center, Inc., 1986). (May 30, 1st edition issued, see pages 77-100). Examples of such methods include acquisition of auxotrophic mutants, acquisition of L-amino acid analog-resistant strains, acquisition of metabolic control mutants, and recombination with enhanced activity of L-amino acid biosynthetic enzymes. The creation of stocks. In the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation that are imparted may be single, or two or more. In addition, L-amino acid biosynthetic enzymes whose activities are enhanced in breeding L-amino acid-producing bacteria may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with enhancing the activity of biosynthetic enzymes.

An auxotrophic mutant, an analog resistant strain, or a metabolically controlled mutant having L-amino acid production ability is subjected to normal mutation treatment of the parent strain or wild strain, and the auxotrophic, analog It can be obtained by selecting those exhibiting resistance or metabolic control mutations and having the ability to produce L-amino acids. Normal mutation treatments include X-ray and ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), etc. Treatment with a mutagen is included.

Also, the L-amino acid-producing ability can be imparted or enhanced by enhancing the activity of an enzyme involved in the target L-amino acid biosynthesis. Enhancing enzyme activity can be performed, for example, by modifying bacteria so that expression of a gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like. A detailed method for enhancing the enzyme activity will be described later.

Furthermore, the L-amino acid-producing ability can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. It can be carried out. As used herein, “an enzyme that catalyzes a reaction that produces a compound other than the target L-amino acid by branching from the biosynthetic pathway of the target L-amino acid” includes enzymes involved in the degradation of the target amino acid. It is. A method for reducing the enzyme activity will be described later.

Specific examples of L-amino acid-producing bacteria and methods for imparting or enhancing L-amino acid-producing ability are given below. In addition, any of the modifications exemplified below for imparting or enhancing the properties of L-amino acid-producing bacteria and L-amino acid-producing ability may be used alone or in appropriate combination.

<L-glutamic acid producing bacteria>
Examples of the method for imparting or enhancing L-glutamic acid-producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-glutamic acid biosynthetic enzymes is increased. . Examples of such enzymes include, but are not limited to, glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltBD), isocitrate dehydrogenase (icdA), aconite hydratase (acnA, acnB), citrate synthase (GltA), methyl citrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate Synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bis Acid aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase ( eda), transhydrogenase. The parentheses are abbreviations for genes encoding the enzymes (the same applies to the following description). Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase.

Strains belonging to the family Enterobacteriaceae that have been modified to increase expression of the citrate synthase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A, and EP952221A Can be mentioned. Examples of strains belonging to the family Enterobacteriaceae that have been modified to increase the expression of the Entner-Doudoroff pathway genes (edd, eda) include those disclosed in EP1352966B.

The method for imparting or enhancing the ability to produce L-glutamic acid is, for example, selected from enzymes that catalyze a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid. Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned. Examples of such enzymes include, but are not limited to, isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxyacid synthase (ilvG), Acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB), succinate dehydrogenase (sdhABCD), 1-pyrroline-5-carboxylate dehydrogenase ( putA). Among these enzymes, for example, it is preferable to reduce or eliminate α-ketoglutarate dehydrogenase activity.

Escherichia bacteria with reduced or deficient α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. In addition, a method for reducing or eliminating α-ketoglutarate dehydrogenase activity in enteric bacteria such as Pantoea bacteria, Enterobacter bacteria, Klebsiella bacteria, Erwinia bacteria, and the like are disclosed in U.S. Patent No. 6,197,559, U.S. Patent No. 6,682,912, This is disclosed in US Pat. No. 6,331,419, US Pat. No. 8,129,151, and WO2008 / 075483. Specific examples of bacteria belonging to the genus Escherichia with reduced or deficient α-ketoglutarate dehydrogenase activity include the following strains.
E. coli W3110sucA :: Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA :: Kmr is a strain obtained by disrupting the sucA gene encoding the α-ketoglutarate dehydrogenase of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase activity.

In addition, L-glutamic acid-producing bacteria or parent strains for inducing them include Pantoea ananatis AJ13355 strain (FERM BP-6614), Pantoea ananatis SC17 strain (FERM BP-11091), Pantoea ananatis SC17 (0) strain (VKPM B) -9246) and the like. The AJ13355 strain is a strain isolated as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH from soil in Iwata City, Shizuoka Prefecture. The SC17 strain is a strain selected from the AJ13355 strain as a low mucus production mutant (US Pat. No. 6,596,517). On February 4, 2009, SC17 shares were incorporated by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently the National Institute of Technology and Evaluation, Patent Biological Depositary Center, ZIP Code: 292-0818, Address: Japan Kazusa Kamashizu 2-5-8-5120, Kisarazu City, Chiba Prefecture, Japan), and has been given the accession number FERM BP-11091. AJ13355 shares were founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Depositary Center, Postal Code: 292-0818, Address: Chiba, Japan Deposited in Kazusa, Kazusa, Kazusa 2-5-8 120) under the deposit number FERM P-16644, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6614 Has been.

In addition, examples of L-glutamic acid-producing bacteria and parent strains for inducing them also include Pantoea bacteria with reduced or deficient α-ketoglutarate dehydrogenase activity. Such strains include the AJ13356 strain (US Pat. No. 6,331,419) which is the E1 subunit gene (sucA) deficient strain of the α-ketoglutarate dehydrogenase of the AJ13355 strain, and the SC17sucA strain which is the sucA gene deficient strain of the SC17 strain ( US Pat. No. 6,596,517). The AJ13356 strain was founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Depositary Center, Postal Code: 292-0818, Address: Chiba, Japan. Deposited at Kisarazu City Kazusa Kamashichi 2-5-8 120) under the accession number FERM P-16645 and transferred to the international deposit under the Budapest Treaty on 11 January 1999 and given the accession number FERM BP-6616 ing. The SC17sucA strain was also assigned the private number AJ417. On February 26, 2004, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (now the National Institute for Product Evaluation Technology Patent Biological Deposit Center, postal code) : 292-0818, Address: Kazusa Kamashitsu 2-5-8 津 120, Kisarazu, Chiba, Japan), deposited under the accession number FERM BP-8646.

The AJ13355 strain was identified as Enterobacter agglomerans at the time of its isolation, but has recently been reclassified as Pantoea anaananatis by 16S rRNA nucleotide sequence analysis and the like. Therefore, the AJ13355 strain and the AJ13356 strain are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification.

Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include Pantoea bacteria such as Pantoea ananatis SC17sucA / RSFCPG + pSTVCB strain, Pantoea ananatis AJ13601 strain, Pantoea ananatis NP106 strain, and Pantoea ananatis NA1 strain . The SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppc), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, and Brevi This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were registered with the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Biological Depositary Center, Postal Code: 292-0818, Address: Chiba, Japan. Deposited at Kisarazu City Kazusa Kamashika 2-5-8 120) under the accession number FERM P-17516, transferred to an international deposit based on the Budapest Treaty on July 6, 2000 and given the accession number FERM BP-7207 ing.

Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include strains in which both α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deficient (JP 2010) -041920). Specific examples of such a strain include a pantoeaPananatis NA1 sucAsdhA double-deficient strain (Japanese Patent Laid-Open No. 2010-041920).

In addition, examples of L-glutamic acid-producing bacteria or parent strains for inducing them include auxotrophic mutants. Specific examples of the auxotrophic mutant include E. coli VL334thrC ⁺ (VKPM B-8961) (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph having a mutation in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). E. coli VL334thrC ⁺ is an L-isoleucine-requiring L-glutamic acid-producing bacterium obtained by introducing a wild type allele of the thrC gene into VL334. The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K-12 strain (VKPM B-7).

In addition, examples of L-glutamic acid-producing bacteria or parent strains for inducing them also include strains resistant to aspartic acid analogs. These strains may be deficient in α-ketoglutarate dehydrogenase activity, for example. Specific examples of strains resistant to aspartate analogs and lacking α-ketoglutarate dehydrogenase activity include, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5,908,768), and L-glutamic acid. E. coli FFRM P-12379 (US Pat. No. 5,393,671) and E. coli AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) are known.

As a method for imparting or enhancing L-glutamic acid-producing ability, for example, a bacterium is modified so that the activity of D-xylulose-5-phosphate-phosphoketolase and / or fructose-6-phosphate phosphoketolase is increased. There is also a method to do (Special Table 2008-509661). Either one or both of D-xylulose-5-phosphate-phosphoketolase activity and fructose-6-phosphate phosphoketolase activity may be enhanced. In the present specification, D-xylulose-5-phosphate phosphoketolase and fructose-6-phosphate phosphoketolase may be collectively referred to as phosphoketolase.

D-xylulose-5-phosphate-phosphoketolase activity is the consumption of phosphoric acid to convert xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl phosphate, and one molecule of H ₂ O Means the activity of releasing. This activity is measured by the method described in Goldberg, M. et al. (Methods Enzymol., 9,515-520 (1966)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.

In addition, fructose-6-phosphate phosphoketolase activity means that phosphoric acid is consumed, fructose 6-phosphate is converted into erythrose-4-phosphate and acetyl phosphate, and one molecule of H ₂ O is released. Means activity. This activity is measured by the method described in Racker, E (Methods Enzymol., 5, 276-280 (1962)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.

Examples of a method for imparting or enhancing L-glutamic acid producing ability include, for example, enhancing expression of yhfK gene (WO2005 / 085419) and ybjL gene (WO2008 / 133161) which are L-glutamic acid excretion genes. It is done.

<L-glutamine producing bacteria>
Examples of the method for imparting or enhancing L-glutamine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-glutamine biosynthesis enzymes is increased. . Examples of such an enzyme include, but are not limited to, glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA). The activity of glutamine synthetase may be enhanced by disrupting the glutamine adenyltransferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121).

The method for imparting or enhancing L-glutamine production ability is, for example, selected from an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamine to produce a compound other than L-glutamine. Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned. Such an enzyme is not particularly limited, and includes glutaminase.

As an L-glutamine producing bacterium or a parent strain for deriving the same, specifically, for example, Escherichia having a mutant glutamine synthetase in which the tyrosine residue at position 397 of glutamine synthetase is substituted with another amino acid residue Strains belonging to the genus (US Patent Application Publication No. 2003-0148474).

<L-proline producing bacteria>
Examples of the method for imparting or enhancing L-proline production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-proline biosynthesis enzymes is increased. . Such enzymes include glutamate-5-kinase (proB), γ-glutamyl-phosphate reductase, pyrroline-5-carboxylate reductase (putA). For the enhancement of the enzyme activity, for example, the proB gene (German Patent No. 3127361) encoding glutamate-5-kinase in which feedback inhibition by L-proline is released can be suitably used.

In addition, as a method for imparting or enhancing L-proline production ability, for example, a method of modifying bacteria so that the activity of an enzyme involved in L-proline degradation is reduced. Examples of such an enzyme include proline dehydrogenase and ornithine aminotransferase.

Specific examples of L-proline-producing bacteria or parent strains for deriving them include, for example, E. coli NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), E. coli VKPM B-8012 ( Russian patent application 2000124295), E. coli plasmid variant described in German Patent 3127361, Bloom FR et al (The 15th Miami winter symposium, 1983, p.34), E. coli plasmid variant, 3, E. coli 702 strain (VKPMB-8011) resistant to 4-dehydroxyproline and azatidine-2-carboxylate, E. coli 702ilvA strain (VKPM B-8012) (EP 1172433) which is a 702 ilvA gene-deficient strain Is mentioned.

<L-threonine producing bacteria>
Examples of the method for imparting or enhancing the ability to produce L-threonine include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-threonine biosynthetic enzymes is increased. . Examples of such enzymes include, but are not limited to, aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA), homoserine kinase (thrB), threonine synthase ( threonine synthase) (thrC), aspartate aminotransferase (aspartate transaminase) (aspC). Among these enzymes, it enhances the activity of one or more enzymes selected from aspartokinase III, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase. Is preferred. The L-threonine biosynthesis gene may be introduced into a strain in which threonine degradation is suppressed. Examples of strains in which threonine degradation is suppressed include E. coli TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).

The activity of the L-threonine biosynthetic enzyme is inhibited by the final product L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is preferable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute a threonine operon, and the threonine operon forms an attenuator structure. Expression of the threonine operon is inhibited by isoleucine and threonine in the culture medium, and is suppressed by attenuation. Enhanced expression of the threonine operon can be achieved by removing the leader sequence or attenuator in the attenuation region (Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M., Gumport, R. I., and Gardner, J. F. J. Mol. Biol. 194: 59-69 1987 (1987); WO02 / 26993; WO2005 / 049808; WO2003 / 097839).

A unique promoter exists upstream of the threonine operon, but this promoter may be replaced with a non-natural promoter (see pamphlet of WO98 / 04715). In addition, the threonine operon may be constructed so that a gene involved in threonine biosynthesis is expressed under the control of a lambda phage repressor and promoter (see European Patent No. 0593792). Bacteria modified so as not to be subjected to feedback inhibition by L-threonine can also be obtained by selecting a strain resistant to α-amino-β-hydroxyvaleric acid (AHV), which is an L-threonine analog.

Thus, the threonine operon modified so as not to be subjected to feedback inhibition by L-threonine is improved in the expression level in the host by increasing the copy number or being linked to a strong promoter. Is preferred. An increase in copy number can be achieved by introducing a plasmid containing a threonine operon into the host. An increase in copy number can also be achieved by transferring the threonine operon onto the host genome using a transposon, Mu phage, or the like.

In addition, examples of a method for imparting or enhancing L-threonine production ability include a method for imparting L-threonine resistance to a host and a method for imparting L-homoserine resistance. The imparting of resistance can be achieved, for example, by enhancing the expression of a gene that imparts resistance to L-threonine or a gene that imparts resistance to L-homoserine. Examples of genes that confer resistance include rhtA gene (Res. Microbiol. 154: 123-135 (2003)), rhtB gene (European Patent Application Publication No. 0994190), rhtC gene (European Patent Application Publication No. 1013765) ), YfiK gene, and yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

Specific examples of L-threonine-producing bacteria or parent strains for deriving them include, for example, E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Patent No. 5,175,107, US Patent No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Patent No. 5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 ( EP 1149911 A), and E. coli VKPM B-5318 (EP 0593792 B).

VKPM B-3996 strain is a strain obtained by introducing plasmid pVIC40 into TDH-6 strain. The TDH-6 strain is sucrose-assimilating, lacks the thrC gene, and has a leaky mutation in the ilvA gene. The VKPM B-3996 strain has a mutation that imparts resistance to a high concentration of threonine or homoserine in the rhtA gene. The plasmid pVIC40 is a plasmid in which a thrA ^* BC operon containing a mutant thrA gene encoding an aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine and a wild type thrBC gene is inserted into an RSF1010-derived vector (US Patent) No. 5,705,371). This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I substantially desensitized to feedback inhibition by threonine. B-3996 was deposited on 19 November 1987 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) with accession number RIA 1867. . This stock was also assigned to the Lucian National Collection of Industrial Microorganisms (VKPM) on April 7, 1987, under the accession number VKPM (FGUP GosNII Genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russia). Deposited at B-3996.

The strain VKPM B-5318 is non-isoleucine-requiring and retains the plasmid pPRT614 in which the control region of the threonine operon in the plasmid pVIC40 is replaced with a temperature-sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318 Has been deposited internationally.

The thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990). The thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12. The thrB gene encoding homoserine kinase of Escherichia coli has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 49175990). The thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12. The thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. In addition, a thrA * BC operon containing a mutant thrA gene encoding an aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine and a wild type thrBC gene is known in the threonine-producing strain E. coli VKPM B-3996. It can be obtained from plasmid pVIC40 (US Pat. No. 5,705,371).

The rhtA gene of E. coli is present at 18 minutes of the E. coli chromosome close to the glnHPQ operon, which encodes a glutamine transport system element. The rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant toosehomoserine andeonthreonine (resistant to homoserine and threonine)). It has also been found that the rhtA23 mutation conferring resistance to high concentrations of threonine or homoserine is a G → A substitution at position -1 relative to the ATG initiation codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).

The asd gene of E. coli has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene ( White, TJ et al., Trends Genet., 5, 185 (1989)). The asd gene of other microorganisms can be obtained similarly.

In addition, the aspC gene of E. 既 に coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895), and obtained by PCR using a primer prepared based on the nucleotide sequence of the gene be able to. The aspC gene of other microorganisms can be obtained similarly.

<L-lysine producing bacteria>
Examples of a method for imparting or enhancing L-lysine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-lysine biosynthesis enzymes is increased. . Such enzymes include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate Deaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat. No. 6,040,160), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (aspartate semialdehyde dehydrogenase) ) (Asd), aspartate aminotransferase (aspartate transaminase) (aspC), diaminopimelate epi Diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl-diaminopimelate deacylase (dapE), and aspartase (aspA) (195) ). Among these enzymes, for example, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and Preferably, the activity of one or more enzymes selected from succinyl diaminopimelate deacylase is enhanced. In addition, in L-lysine producing bacteria or a parent strain for deriving the same, a gene (cyo) (EP 1170376 A) involved in energy efficiency, a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) ( US Pat. No. 5,830,716), ybjE gene (WO2005 / 073390), or combinations thereof may have increased expression levels. Aspartokinase III (lysC) is subject to feedback inhibition by L-lysine. To enhance the activity of the enzyme, a mutant lysC gene encoding aspartokinase III that has been desensitized to feedback inhibition by L-lysine is used. It may be used (US Pat. No. 5,932,453). In addition, since it is subjected to feedback inhibition by dihydrodipicolinate synthase (dapA) L-lysine, in order to enhance the activity of the enzyme, a mutant type encoding dihydrodipicolinate synthase from which feedback inhibition by L-lysine is released The dapA gene may be used.

The method for imparting or enhancing L-lysine production ability is, for example, selected from enzymes that catalyze the reaction of branching from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine. Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned. Such enzymes include, but are not limited to, homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malic enzyme (WO2005 / 010175). .

In addition, examples of L-lysine-producing bacteria or parent strains for inducing them include mutants having resistance to L-lysine analogs. L-lysine analogs inhibit the growth of bacteria such as Enterobacteriaceae and coryneform bacteria, but this inhibition is completely or partially released when L-lysine is present in the medium. The L-lysine analog is not particularly limited, and examples thereof include oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, and α-chlorocaprolactam. Mutant strains having resistance to these lysine analogs can be obtained by subjecting bacteria to normal artificial mutation treatment.

Specific examples of L-lysine-producing bacteria or parent strains for deriving them include, for example, E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) and E. coli VL611. Can be mentioned. In these strains, feedback inhibition of aspartokinase by L-lysine is released. Specific examples of L-lysine-producing bacteria or parent strains for inducing them include E. 例 え ば coli AJIK01 strain (NITE BP-01520). The AJIK01 strain was named E. coli AJ111046. On January 29, 2013, the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Postal Code: 292-0818, Address: Kazusa Kama, Kisarazu City, Chiba Prefecture, Japan) No. 2-5-8 (120)), transferred to an international deposit based on the Budapest Treaty on May 15, 2014, and assigned the deposit number NITE BP-01520.

Specific examples of L-lysine-producing bacteria or parent strains for inducing them include E. coli WC196 strain. The WC196 strain was bred by conferring AEC resistance to the W3110 strain derived from E. coli K-12 (US Pat. No. 5,827,698). The WC196 strain was named E. coli AJ13069, and on December 6, 1994, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Depositary Center, ZIP Code: 292- 0818, address: 2-5-8 鎌 120, Kazusa Kamashitsu, Kisarazu City, Chiba, Japan), deposited under the accession number FERM P-14690, transferred to an international deposit based on the Budapest Treaty on September 29, 1995. No. FERM BP-5252 (US Pat. No. 5,827,698).

Preferred L-lysine producing bacteria include E.coli WC196ΔcadAΔldc and E.coli WC196ΔcadAΔldc / pCABD2 (WO2010 / 061890). WC196ΔcadAΔldc is a strain constructed by disrupting the cadA and ldcC genes encoding lysine decarboxylase from the WC196 strain. WC196ΔcadAΔldc / pCABD2 is a strain constructed by introducing plasmid pCABD2 (US Pat. No. 6,040,160) containing a lysine biosynthesis gene into WC196ΔcadAΔldc. WC196ΔcadAΔldc was named AJ110692, and on October 7, 2008, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently, National Institute of Technology and Evaluation, Patent Biological Depositary Center, ZIP Code: 292-0818, Address: Room 2-5-8 120, Kazusa Kamashitsu, Kisarazu, Chiba, Japan) was deposited internationally under the deposit number FERM BP-11027. pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes.

<L-arginine producing bacteria>
Examples of the method for imparting or enhancing L-arginine-producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-arginine biosynthesis enzymes is increased. . Examples of such enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine Examples include transaminase (argD), acetylornithine deacetylase (argE) ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthase (carAB). Examples of the N-acetylglutamate synthase (argA) gene include mutant N-acetylglutamate synthase in which amino acid residues corresponding to the 15th to 19th positions of the wild type are substituted and feedback inhibition by L-arginine is released. It is preferable to use a gene to be encoded (European Application Publication No. 1170361).

Specific examples of L-arginine-producing bacteria or parent strains for inducing the same include, for example, E. coli 237 strain (VKPM B-7925) (US Patent Application Publication 2002/058315 A1), mutant N-acetylglutamic acid Its derivative strain さ れ (Russian patent application No. 2001112869, EP1170361A1) introduced with the argA gene encoding synthase, E.237coli 382 strain (VKPM B-7926) 237 (VKPM B-7926) EP1170358A1) and E. coli 382ilvA + strain, which is a strain in which the wild-type ilvA gene derived from E. coli K-12 strain is introduced into 382 strain. E. coli 237 shares were registered with VKPM B-7925 on April 10, 2000 at Lucian National Collection of Industrial Microorganisms (1 Dorozhny proezd., 1 Moscow 117545, Russia) And was transferred to an international deposit under the Budapest Treaty on May 18, 2001. E. coli 382 shares were awarded VKPM B-7926 to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on April 10, 2000 Deposited at

In addition, examples of L-arginine-producing bacteria or parent strains for inducing them include strains having resistance to amino acid analogs and the like. Such strains include, for example, α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S- (2-aminoethyl) -cysteine, α-methylserine, β-2-thienylalanine, or Examples include Escherichia coli mutants having resistance to sulfaguanidine (see JP-A-56-106598).

<L-citrulline-producing bacteria and L-ornithine-producing bacteria>
L-citrulline and L-ornithine share a biosynthetic pathway with L-arginine. Thus, N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), and / or acetylornithine By increasing the enzyme activity of deacetylase (argE), the ability to produce L-citrulline and / or L-ornithine can be imparted or enhanced (WO 2006-35831).

<L-histidine producing bacteria>
Examples of the method for imparting or enhancing L-histidine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-histidine biosynthesis enzymes is increased. . Examples of such an enzyme include, but are not limited to, ATP phosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisI), phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleoside. Examples thereof include tide isomerase (hisA), amide transferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), and histidinol dehydrogenase (hisD).

Of these, L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine. Therefore, the ability to produce L-histidine can be imparted or enhanced, for example, by introducing a mutation that confers resistance to feedback inhibition in the ATP phosphoribosyltransferase gene (hisG) (Russian Patent No. 2003677 and No. 2). 2119536).

Specific examples of L-histidine-producing bacteria or parent strains for deriving them include, for example, E. coli 24 strain (VKPM B-5945, RU2003677), E. coli NRRL B-12116-B12121 (US Patent No. 4,388,405). No.), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347), E. coli H-9341 (FERM BP-6674) (EP1085087), E Coli AI80 / pFM201 (U.S. Patent No. 6,258,554), E. coli FERM P-5038 and 5048 (Japanese Patent Laid-Open No. 56-005099) into which a vector holding a DNA encoding an L-histidine biosynthetic enzyme was introduced E. coli strain 導入 (EP1016710A) introduced with a gene for amino acid transport, E. coli 80 strain (VKPM B-7270) with resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin , (Russian Patent No. 2119536) and other strains belonging to the genus Escherichia.

<L-cysteine producing bacteria>
Examples of the method for imparting or enhancing L-cysteine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-cysteine biosynthesis enzymes is increased. . Examples of such an enzyme include, but are not limited to, serine acetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase (serA). Serine acetyltransferase activity can be enhanced, for example, by introducing a mutant cysE gene encoding a mutant serine acetyltransferase resistant to feedback inhibition by cysteine into bacteria. Mutant serine acetyltransferases are disclosed, for example, in JP-A-11-155571 and US Patent Publication No. 20050112731. Further, the 3-phosphoglycerate dehydrogenase activity can be enhanced by introducing, for example, a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine into a bacterium. Mutant 3-phosphoglycerate dehydrogenase is disclosed, for example, in US Pat. No. 6,180,373.

The method for imparting or enhancing L-cysteine production ability is selected from, for example, an enzyme that catalyzes a reaction that branches from the biosynthesis pathway of L-cysteine to produce a compound other than L-cysteine. Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned. Examples of such enzymes include enzymes involved in the degradation of L-cysteine. The enzyme involved in the degradation of L-cysteine is not particularly limited, but cystathionine-β-lyase (metC) (Japanese Patent Laid-Open No. 11-155571, Chandra et. Al., Biochemistry, 21 (1982) 3064-3069)) Tryptophanase (tnaA) (Japanese Patent Laid-Open No. 2003-169668, Austin 、 Newton et. Al., J. Biol. Chem. 240 (1965) 1211-1218), O-acetylserine sulfhydrylase B (cysM) (special JP 2005-245311), malY gene product (JP 2005-245311), d0191 gene product of Pantoea 遺 伝 子 ananatis (JP 2009-232844).

In addition, examples of methods for imparting or enhancing L-cysteine production ability include enhancing the L-cysteine excretion system and enhancing the sulfate / thiosulfate transport system. Examples of proteins of the L-cysteine excretion system include proteins encoded by the ydeD gene (JP 2002-233384), proteins encoded by the yfiK gene (JP 2004-49237), emrAB, emrKY, yojIH, acrEF, bcr, And each protein encoded by each gene of cusA (Japanese Patent Laid-Open No. 2005-287333), and protein encoded by the yeaS gene (Japanese Patent Laid-Open No. 2010-187552). Examples of the sulfate / thiosulfate transport system protein include proteins encoded by the cysPTWAM gene cluster.

Specific examples of L-cysteine-producing bacteria or parent strains for deriving them include, for example, E. coli JM15 (US Patent) transformed with various cysE alleles encoding mutant serine acetyltransferase resistant to feedback inhibition. No. 6,218,168, Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663), cysteine desulfhydrase, which has an overexpressed gene encoding a protein suitable for excretion of substances toxic to cells Examples include E. coli strain (JP11155571A2) with reduced activity and E. coli W3110 (WO01 / 27307A1) with increased activity of the transcriptional control factor of the positive cysteine regulon encoded by the cysB gene.

<L-methionine producing bacteria>
Examples of L-methionine-producing bacteria or parent strains for inducing them include L-threonine-requiring strains and mutants having resistance to norleucine (Japanese Patent Laid-Open No. 2000-139471). In addition, examples of L-methionine-producing bacteria or parent strains for deriving them also include strains that retain mutant homoserine transsuccinylase that is resistant to feedback inhibition by L-methionine (Japanese Patent Laid-Open No. 2000-139471). , US20090029424). Since L-methionine is biosynthesized with L-cysteine as an intermediate, L-methionine production ability can be improved by improving L-cysteine production ability (Japanese Patent Laid-Open No. 2000-139471, US20080311632).

Specific examples of L-methionine-producing bacteria or parent strains for inducing them include, for example, E. coli AJ11539 (NRRL B-12399), E. coli AJ11540 (NRRL B-12400), E. coli AJ11541 (NRRL B-12401), E. coli AJ11542 (NRRL B-12402) (British Patent No. 2075055), E. coli 218 strain (VKPM B-8125) having resistance to norleucine, an analog of L-methionine (Russian Patent No. 2209248) No.), 73 shares (VKPM B-8126) (Russian Patent No. 2215782), E. coli AJ13425 (FERM P-16808) (Japanese Patent Laid-Open No. 2000-139471). The AJ13425 strain lacks a methionine repressor, weakens intracellular S-adenosylmethionine synthetase activity, and produces intracellular homoserine transsuccinylase activity, cystathionine γ-synthase activity, and aspartokinase-homoserine dehydrogenase II. L-threonine-requiring strain derived from E. coli W3110 with enhanced activity.

<L-leucine producing bacteria>
Examples of the method for imparting or enhancing the ability to produce L-leucine include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-leucine biosynthesis enzymes is increased. . Examples of such an enzyme include, but are not limited to, an enzyme encoded by a gene of leuABCD operon. For enhancing enzyme activity, for example, a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase from which feedback inhibition by L-leucine has been released can be suitably used.

Specific examples of L-leucine-producing bacteria or parent strains for inducing the same include, for example, leucine-resistant E. coli strains (eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)), β- E. coli strains resistant to leucine analogs such as 2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP-B-62-34397 and JP-A-8-70879), WO96 And strains belonging to the genus Escherichia such as E. coli strain and E. coli H-9068 (JP-A-8-70879) obtained by the genetic engineering method described in / 06926.

<L-isoleucine producing bacterium>
Examples of the method for imparting or enhancing L-isoleucine producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-isoleucine biosynthesis enzymes is increased. . Examples of such an enzyme include, but are not limited to, threonine deaminase and acetohydroxy acid synthase (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).

Specific examples of L-isoleucine-producing bacteria or parent strains for inducing them include mutants having resistance to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate, etc. And an Escherichia bacterium such as a mutant strain resistant to DL-ethionine and / or arginine hydroxamate in addition to the isoleucine analog (Japanese Patent Laid-Open No. 5-130882).

<L-valine producing bacteria>
Examples of a method for imparting or enhancing L-valine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-valine biosynthetic enzymes is increased. . Examples of such enzymes include, but are not limited to, enzymes encoded by genes of ilvGMEDA operon and ilvBNC operon. ilvBN encodes acetohydroxy acid synthase, and ilvC encodes isomeroreductase (WO 00/50624). The ilvGMEDA operon and the ilvBNC operon are subject to expression suppression (attenuation) by L-valine, L-isoleucine, and / or L-leucine. Therefore, in order to enhance the enzyme activity, it is preferable to remove or modify the region necessary for attenuation and to cancel the expression suppression by the produced L-valine. The threonine deaminase encoded by the ilvA gene is an enzyme that catalyzes the deamination reaction from L-threonine to 2-ketobutyric acid, which is the rate-limiting step of the L-isoleucine biosynthesis system. Therefore, for L-valine production, it is preferable that the ilvA gene is disrupted and the threonine deaminase activity is reduced.

The method for imparting or enhancing L-valine-producing ability is, for example, selected from enzymes that catalyze a reaction that branches from the biosynthetic pathway of L-valine to produce a compound other than L-valine. Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned. Examples of such enzymes include, but are not limited to, threonine dehydratase involved in L-leucine synthesis and enzymes involved in D-pantothenic acid synthesis (International Publication No. 00/50624).

Specific examples of the L-valine-producing bacterium or the parent strain for deriving the same include, for example, the E. coli strain (US Pat. No. 5,998,178) that has been modified to overexpress the ilvGMEDA operon.

In addition, examples of L-valine-producing bacteria and parent strains for deriving the same also include strains having mutations in aminoacyl t-RNA synthetase (US Pat. No. 5,658,766). Examples of such a strain include E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase. E. coli VL1970 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on June 24, 1988 under the accession number VKPM B-4411. It has been deposited. In addition, examples of L-valine-producing bacteria or parent strains for deriving the same also include mutant strains (WO96 / 06926) that require lipoic acid for growth and / or lack H ⁺ -ATPase. .

<L-tryptophan producing bacteria, L-phenylalanine producing bacteria, L-tyrosine producing bacteria>
Examples of methods for imparting or enhancing L-tryptophan production ability, L-phenylalanine production ability, and / or L-tyrosine production ability include biosynthesis of L-tryptophan, L-phenylalanine, and / or L-tyrosine. Examples include a method of modifying a bacterium so that the activity of one or more enzymes selected from system enzymes is increased.

Biosynthetic enzymes common to these aromatic amino acids are not particularly limited, but 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase (aroG), 3-dehydroquinate synthase (aroB) Shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolic acid pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC) (European Patent No. 763127). Expression of genes encoding these enzymes is controlled by a tyrosine repressor (tyrR), and the activity of these enzymes may be enhanced by deleting the tyrR gene (European Patent No. 763127).

Examples of the L-tryptophan biosynthesis enzyme include, but are not limited to, anthranilate synthase (trpE), tryptophan synthase (trpAB), and phosphoglycerate dehydrogenase (serA). For example, L-tryptophan production ability can be imparted or enhanced by introducing DNA containing a tryptophan operon. Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Since anthranilate synthase is subject to feedback inhibition by L-tryptophan, in order to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used. Since phosphoglycerate dehydrogenase is feedback-inhibited by L-serine, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used to enhance the activity of the enzyme. Furthermore, L-tryptophan-producing ability is imparted or enhanced by increasing the expression of an operon consisting of malate synthase (aceB), isocitrate lyase (aceA), and isocitrate dehydrogenase kinase / phosphatase (aceK). (WO2005 / 103275).

The L-phenylalanine biosynthetic enzyme is not particularly limited, and examples thereof include chorismate mutase and prefenate dehydratase. Chorismate mutase and prefenate dehydratase are encoded by the pheA gene as a bifunctional enzyme. Since chorismate mutase-prefenate dehydratase is feedback-inhibited by L-phenylalanine, in order to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.

The L-tyrosine biosynthetic enzyme is not particularly limited, and examples thereof include chorismate mutase and prephenate dehydrogenase. Chorismate mutase and prefenate dehydrogenase are encoded by the tyrA gene as a bifunctional enzyme. Since chorismate mutase-prefenate dehydrogenase is feedback-inhibited by L-tyrosine, to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.

The L-tryptophan, L-phenylalanine, and / or L-tyrosine producing bacterium may be modified so that biosynthesis of aromatic amino acids other than the target aromatic amino acid is lowered. In addition, L-tryptophan, L-phenylalanine, and / or L-tyrosine-producing bacteria may be modified so that the by-product uptake system is enhanced. By-products include aromatic amino acids other than the desired aromatic amino acid. Examples of genes encoding uptake systems of by-products include, for example, uptake systems of tnaB and mtr, which are L-tryptophan uptake systems, and pheP, L-tyrosine, which are genes encoding uptake systems of L-phenylalanine. TyrP, which is a gene coding for (EP1484410).

As an L-tryptophan-producing bacterium or a parent strain for deriving it, specifically, for example, E. coli JP4735 / pMU3028 carrying a mutant trpS gene encoding a partially inactivated tryptophanyl-tRNA synthetase. DSM10122) and JP6015 / pMU91 (DSM10123) (U.S. Patent No. 5,756,345), E. coli SV164 with trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan, phosphoglycerate dehydrogenase not subject to feedback inhibition by serine E. coli SV164 (pGH5) (U.S. Pat.No. 6,180,373) with serA allele encoding and trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan, coding for anthranilate synthase not subject to feedback inhibition by tryptophan A strain introduced with a tryptophan operon containing a trpE allele (JP 57-71397, JP 62-244382, U.S. Pat.No. 4,371,614), E. coliGX AGX17 (pGX44) lacking tryptophanase NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) (U.S. Pat.No. 4,371,614), E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Patent No. No. 6,319,696), strains belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).

As an L-phenylalanine producing bacterium or a parent strain for deriving the same, specifically, for example, E. coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM) lacking chorismate mutase-prefenate dehydrogenase and tyrosine repressor B-8197) (WO03 / 044191), E. coli HW1089 (ATCC 55371) (U.S. Patent No. 5,354,672), carrying a mutant pheA34 gene encoding chorismate mutase-prefenate dehydratase with released feedback inhibition (US Patent No. 5,354,672), E .Coli MWEC 101-b (KR8903681), E.coli NRRL B-12141, NRRL B-12145, NRRL B-12146, NRRL B-12147 (US Pat. No. 4,407,952). Further, as an L-phenylalanine-producing bacterium or a parent strain for deriving the same, specifically, for example, E. coli K-12 that retains a gene encoding chorismate mutase-prefenate dehydratase in which feedback inhibition is released. <W3110 (tyrA) / pPHAB> (FERM BP-3566), E. coli K-12 <W3110 (tyrA) / pPHAD> (FERM BP-12659), E. coli K-12 <W3110 (tyrA) / pPHATerm> (FERM BP-12662), E. coli K-12 AJ 12604 <W3110 (tyrA) / pBR-aroG4, pACMAB> (FERM BP-3579) (EP 488424 B1). Specific examples of L-phenylalanine-producing bacteria or parent strains for inducing them include, for example, strains belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or the yddG gene is increased (US2003 / 0148473, US2003 / 0157667, WO03 / 044192).

In addition, examples of a method for imparting or enhancing L-amino acid-producing ability include a method of modifying a bacterium so that the activity of discharging L-amino acid from the bacterium cell is increased. The activity to excrete L-amino acids can be increased, for example, by increasing the expression of a gene encoding a protein that excretes L-amino acids. Examples of genes encoding proteins that excrete various amino acids include b2682 gene (ygaZ), b2683 gene (ygaH), b1242 gene (ychE), and b3434 gene (yhgN) (Japanese Patent Laid-Open No. 2002-300874) .

In addition, examples of a method for imparting or enhancing L-amino acid producing ability include a method for modifying bacteria so that the activity of a protein involved in sugar metabolism or a protein involved in energy metabolism is increased.

Proteins involved in sugar metabolism include proteins involved in sugar uptake and glycolytic enzymes. Examples of genes encoding proteins involved in sugar metabolism include glucose 6-phosphate isomerase gene (pgi; WO 01/02542 pamphlet), phosphoenolpyruvate synthase gene (pps; EP 877090 specification) , Phosphoenolpyruvate carboxylase gene (ppc; WO 95/06114 pamphlet), pyruvate carboxylase gene (pyc; WO 99/18228 pamphlet, European application 1092776), phosphoglucomutase gene (Pgm; WO 03/04598 pamphlet), fructose diphosphate aldolase gene (pfkB, fbp; WO 03/04664 pamphlet), pyruvate kinase gene (pykF; WO 03/008609 pamphlet), transaldolase Gene (talB; WO03 / 008611 pamphlet), fumarase residue Child (fum; International Publication No. 01/02545 pamphlet), non-PTS sucrose uptake gene gene (csc; European Application Publication No. 149911 pamphlet), sucrose utilization gene (scrAB operon; International Publication No. 90/04636 pamphlet) Can be mentioned.

Examples of genes encoding proteins involved in energy metabolism include a transhydrogenase gene (pntAB; US Pat. No. 5,830,716), a cytochrome bo type oxidase (cyoB; European Patent Application Publication No. 1070376) Is mentioned.

The gene used for breeding the above-mentioned L-amino acid-producing bacteria is not limited to the above-exemplified genes or genes having a known base sequence, as long as it encodes a protein having the original function maintained. There may be. For example, a gene used for breeding an L-amino acid-producing bacterium is an amino acid in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of a known protein. It may be a gene encoding a protein having a sequence. As for gene and protein variants, the descriptions relating to the acpP gene and fabF gene and the protein variants encoded by them can be applied mutatis mutandis.

<1-2> Weakness of acpP-fabF operon The bacterium of the present invention has been modified so that the acpP-fabF operon is weakened. Since the acpP-fabF operon is involved in fatty acid biosynthesis (Non-patent Document 1), when L-amino acid production culture is performed using bacteria in which the acpP-fabF operon has been weakened, L-amino acid is used using an unmodified strain. As compared to the case where production culture is performed, it is estimated that as a result of a decrease in inflow of carbon into the fatty acid biosynthetic pathway, surplus carbon and reducing power are used for L-amino acid production and L-amino acid production is improved. The bacterium of the present invention can be obtained by modifying a bacterium having an L-amino acid-producing ability so that the acpP-fabF operon is weakened. The bacterium of the present invention can also be obtained by imparting or enhancing L-amino acid-producing ability after modifying the bacterium so that the acpP-fabF operon is weakened. In addition, the bacterium of the present invention may have acquired L-amino acid-producing ability by being modified so that the acpP-fabF operon is weakened. The modification for constructing the bacterium of the present invention can be performed in any order.

“AcpP-fabF operon is weakened” means that the activity of the protein encoded by the gene of the acpP-fabF operon is decreased and / or the expression of the gene of the acpP-fabF operon is decreased. . “Gene expression decreases” means that the gene transcription amount (mRNA amount) decreases and / or the gene translation amount (protein amount) decreases. The “gene of the acpP-fabF operon” refers to the acpP gene and / or the fabF gene. That is, the “protein encoded by the gene of the acpP-fabF operon” refers to a protein encoded by the acpP gene and / or a protein encoded by the fabF gene (ie, AcpP protein and / or FabF protein). As described later, the activity of the protein can be achieved, for example, by weakening the expression of the gene encoding the protein or by destroying the gene encoding the protein. That is, “the acpP-fabF operon is weakened” may mean, for example, that the expression of the acpP-fabF operon gene is weakened. In the present invention, for example, the expression of either the acpP gene or the fabF gene may be weakened, or the expression of both may be weakened. That is, the expression of the entire acpP-fabF operon may be weakened.

The acpP gene is a gene encoding an acyl carrier protein (ACP). “ACP” refers to a protein having a function of carrying a fatty acid chain by binding to the fatty acid chain via a 4′-phosphopantethein group during fatty acid biosynthesis. This function is also referred to as “ACP activity”. ACP is translated as inactive apo-ACP, and then 4'-phosphopantethein (4) is added to the serine residue at position 36 of apo-ACP (in the case of Escherichia coli) by ACP synthase. '-phosphopanteheine) is added as a cofactor, resulting in active holo-ACP.

The fabF gene is a gene encoding β-ketoacyl-ACP synthase II. “Β-ketoacyl-ACP synthase II” refers to an enzyme that catalyzes a reaction for producing 3-oxoacyl-ACP (carbon number n + 2) from acyl-ACP (carbon number n) and malonyl-ACP (EC 2.3. 1.41). The activity that catalyzes this reaction is also referred to as “β-ketoacyl-ACP synthase II activity”.

The acpP gene of Escherichia coli K-12 MG1655 strain corresponds to the sequence of 1150838 to 1151074 in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database. The acpP gene of MG1655 strain is synonymous with ECK1080 and JW1080. The AcpP protein of the MG1655 strain is registered as GenBank accession NP_415612 (version NP_415612.1 GI: 16129057, locus_tag = "b1094").

The fabF gene of Escherichia coli K-12 MG1655 strain corresponds to the 1151162 to 1152403 positions in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database. The fabF gene of MG1655 strain is synonymous with ECK1081 and JW1081. The FabF protein of the MG1655 strain is registered as GenBank accession NP_415613 (version NP_415613.1 GI: 16129058, locus_tag = "b1095").

The base sequence of the acpP-fabF operon of MG1655 strain (including upstream 210 bp) is shown in SEQ ID NO: 7. In SEQ ID NO: 7, the base sequence of the acpP gene corresponds to positions 211 to 447, and the base sequence of the fabF gene corresponds to positions 535 to 1776, respectively. In addition, the amino acid sequences of the AcpP protein and FabF protein of the MG1655 strain are shown in SEQ ID NOs: 8 and 9, respectively.

The acpP gene of Pantoea ananatis AJ13355 strain corresponds to the sequence of positions 986154 to 986528 in the genome sequence registered as GenBank accession NC_017531 (VERSION NC_017531.1 GI: 386014600) in the NCBI database. The AcpP protein of AJ13355 strain is registered as GenBank accession YP_005933706 (version YP_005933706.1 GI: 386015425).

The fabF gene of Pantoea ananatis AJ13355 strain corresponds to the sequence of positions 986650 to 987855 in the genome sequence registered as GenBank accession NC_017531 (VERSION NC_017531.1 GI: 386014600) in the NCBI database. In addition, FabF protein of AJ13355 strain is registered as GenBank accession YP_005933707 (version YP_005933707.1 GI: 386015426).

The base sequence of the acpP-fabF operon of AJ13355 strain (including upstream 210 bp) is shown in SEQ ID NO: 10. In SEQ ID NO: 10, the base sequence of the acpP gene corresponds to positions 211 to 585, and the base sequence of the fabF gene corresponds to positions 707 to 1912, respectively. In addition, the amino acid sequences of the AcpP protein and FabF protein of the AJ13355 strain are shown in SEQ ID NOs: 11 and 12, respectively.

The AcpP protein or FabF protein may be a variant of the AcpP protein or FabF protein as long as the original function is maintained. Similarly, the acpP gene or fabF gene may be a variant of the acpP gene or fabF gene as long as the original function is maintained. Such a variant in which the original function is maintained may be referred to as a “conservative variant”. The terms “AcpP protein” or “FabF protein” are intended to encompass, in addition to the AcpP protein or FabF protein, conservative variants thereof, respectively. Similarly, the terms “acpP gene” or “fabF gene” are intended to include conservative variants thereof in addition to the acpP gene or fabF gene, respectively. Examples of the conservative variant include homologues and artificial modifications of the AcpP protein or FabF protein, the acpP gene or the fabF gene.

“The original function is maintained” means that a protein or gene variant has a function (activity or property) corresponding to the function (activity or property) of the original protein or gene. That is, for example, “the original function is maintained” for the AcpP protein means that the protein has ACP activity, and “the original function is maintained” for the FabF protein means that the protein is Having β-ketoacyl-ACP synthase II activity. For example, “the original function is maintained” for the acpP gene means that the gene encodes a protein having ACP activity, and “the original function is maintained” for the fabF gene. Means that the gene encodes a protein having β-ketoacyl-ACP synthase II activity.

The gene encoding the homologue of the AcpP protein or FabF protein can be easily obtained from a public database by BLAST search or FASTA search using the base sequence of the acpP gene or fabF gene as a query sequence, for example. Further, the gene encoding the homologue of the AcpP protein or FabF protein is obtained by PCR using, for example, a chromosome of an organism such as a bacterium as a template and oligonucleotides prepared based on these known gene sequences as primers. be able to.

The acpP gene or fabF gene may be a gene encoding a conservative variant of the AcpP protein or FabF protein. For example, the acpP gene or the fabF gene has one or several amino acids in the amino acid sequence (for example, the amino acid sequence of SEQ ID NO: 8, 9, 11, or 12) as long as it encodes a protein having the original function maintained. It may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at the position are substituted, deleted, inserted, or added. In this case, the corresponding activity (ACP activity or β-ketoacyl-ACP synthase II activity) is usually 70% or more relative to the protein before 1 or several amino acids are substituted, deleted, inserted or added. Preferably, 80% or more, more preferably 90% or more can be maintained. The “one or several” is different depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically 1 to 50, 1 to 40, 1 to 30 It means 1 to 20, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

The substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains the protein function normally. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the organism from which the gene is derived. Also included by

Furthermore, the gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99%, based on the entire amino acid sequence. It may be a gene encoding a protein having a homology of at least% and maintaining the original function. In the present specification, “homology” means “identity”.

The acpP gene or fabF gene is a probe that can be prepared from a known gene sequence, for example, the above base sequence (for example, positions 211 to 447 of SEQ ID NO: 7, positions 535 to 1776 of SEQ ID NO: 7, 211 to DNA that encodes a protein that hybridizes under stringent conditions with a complementary sequence to the whole or part of position 585 or the nucleotide sequence of positions 707 to 1912 of SEQ ID NO: 10 and maintains the original function. Also good. The acpP-fabF operon is a probe that can be prepared from a known gene sequence, for example, the above base sequence (for example, the entire SEQ ID NO: 7, positions 211 to 1776 of SEQ ID NO: 7, the entire SEQ ID NO: 10, or SEQ ID NO: 10 DNA that encodes a protein that hybridizes under stringent conditions with a complementary sequence to the whole or a part of the base sequence at positions 211 to 1912) and maintains the original function. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more between DNAs having homology. Is hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC And 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, salt concentration and temperature corresponding to 0.1% SDS, and conditions of washing once, preferably 2 to 3 times.

As described above, the probe used for the hybridization may be a part of a gene complementary sequence. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, as the probe, a DNA fragment having a length of about 300 bp can be used. When a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

Further, the acpP gene or fabF gene may be one in which any codon is replaced with an equivalent codon as long as it encodes a protein having the original function maintained. For example, the acpP gene or the fabF gene may be modified to have an optimal codon according to the codon usage frequency of the host to be used.

In addition, the description regarding the said gene and protein conservative variant is applicable mutatis mutandis to arbitrary proteins, such as L-amino acid biosynthesis enzyme, and the gene which codes them.

<1-3> Technique for Reducing Protein Activity A technique for reducing the activity of proteins such as AcpP protein and FabF protein will be described below.

“Protein activity decreases” means that the activity per cell of the protein is decreased compared to wild-type strains and parental unmodified strains, and the activity is completely lost. including. Specifically, “the activity of the protein is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. In other words, “activity” in the case of “decrease in protein activity” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost. The activity of the protein is not particularly limited as long as it is lower than that of the non-modified strain. For example, it is 90% or less, 80% or less, 70% or less, 60% or less, 55% compared to the non-modified strain. Hereinafter, it may be reduced to 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0%.

The modification that reduces the activity of the protein is achieved, for example, by reducing the expression of a gene encoding the protein. “Gene expression decreases” includes the case where the gene is not expressed at all. In addition, “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”. The expression of the gene is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 55% or less, 50% or less, 30% or less, 20% or less, 10% or less as compared with the unmodified strain It may be reduced to 5% or less, or 0%.

For example, in E. coli, the acpP gene is known to be essential. Therefore, when reducing the activity of the AcpP protein, if necessary, when the bacterium of the present invention is cultured in a medium, the bacterium of the present invention can proliferate and the desired L-amino acid is produced. The activity remains. That is, the activity of AcpP protein is not reduced to 0% (not completely disappeared) as compared to the unmodified strain. For example, the activity of AcpP protein remains 1% or more, 5% or more, 10% or more, 15% or more, 17% or more, 20% or more, 30% or more, or 50% or more compared to the unmodified strain. It's okay. Specifically, the activity of the AcpP protein is, for example, 1% to 90%, 5% to 80%, 10% to 70%, 15% to 60%, or 17% to 55 compared to the unmodified strain. % May be reduced. In addition, the expression level of the acpP gene is not reduced to 0% compared to the unmodified strain. For example, the expression level of the acpP gene remains 1% or more, 5% or more, 10% or more, 20% or more, 15% or more, 17% or more, 30% or more, or 50% or more compared to the unmodified strain. You can do it. Specifically, the expression level of the acpP gene is, for example, 1% to 90%, 5% to 80%, 10% to 70%, 15% to 60%, or 17% to 17% compared to the unmodified strain. It may be reduced to 55%. Such a description of reducing the activity of the AcpP protein may be applied mutatis mutandis to reducing the activity of the FabF protein.

The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. For example, gene expression can be reduced by altering expression regulatory sequences such as the promoter of the gene, Shine-Dalgarno (SD) sequence (also called ribosome binding site (RBS)), spacer region between RBS and start codon. Can be achieved. AcpP protein is known as an abundant protein in cells, and thus it is suggested that the activity of the wild type promoter of the acpP gene is strong (Non-patent Document 1). Therefore, for example, by replacing the wild-type promoter of the acpP gene with a less active promoter, the expression of the acpP gene can be reduced. Examples of promoters that are less active than the wild type promoter of the acpP gene include the lac promoter and the P _tac84 promoter described in Russian Patent Application Publication No. 2006/134574. In the case of modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted. In addition, reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like. In addition, reduction of gene expression can be achieved, for example, by introducing a mutation that reduces gene expression into the coding region of the gene. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, for example, gene expression itself may be reduced by gene disruption as described below.

The acpP gene and fabF gene are co-transcribed as the acpP-fabF operon. The fabF gene is also transcribed individually from its own promoter. The acpP gene can also be co-transcribed from the fabD gene and the fabG gene in the yceD-rpmF-plsX-fabHDG-acpP-fabF gene cluster. Therefore, for example, the expression of the acpP gene and the fabF gene may be collectively reduced by modifying a promoter that controls co-transcription of the acpP-fabF operon. In addition, for example, the expression of the fabF gene may be decreased alone by modifying the promoter in front of the fabF gene. Further, for example, by modifying the promoter of the fabD gene and / or the fabG gene, the expression of the acpP gene may be decreased together with those genes. In addition, for example, the expression of the acpP gene and / or the fabF gene may be reduced by introducing a mutation that reduces the expression of the gene into the coding region of the acpP gene and / or the fabF gene.

Further, as a mutation that decreases the expression of the acpP gene and / or fabF gene, specifically, for example, a mutation in which cytosine (C) at the position −34 upstream of the translation start point of the acpP gene is replaced with another base. Can be mentioned. The other base is preferably adenine (A).

As used herein, “upstream position 34 of the translation start point of the acpP gene” refers to the position corresponding to position 34 upstream from the start codon (ATG) of the acpP gene in the nucleotide sequence shown in SEQ ID NO: 7. Means. In addition, A of the start codon (ATG) is + 1st, and the upstream side thereof is -1st. In other words, “upstream position 34 of the translation start point of the acpP gene” means the 177th position of the nucleotide sequence shown in SEQ ID NO: 7 (that is, the genome sequence of Escherichia coli K-12 MG1655 strain registered as GenBank accession NC_000913) No. 1150804). “Upstream position 34 of the translation start point of the acpP gene” indicates a relative position with reference to SEQ ID NO: 7, and its absolute position is determined by deletion, insertion, addition, etc. of the base. May go back and forth. That is, “position upstream of position -34 of the translation start point of the acpP gene” is the start codon of the acpP gene when one base is deleted between the base at position 177 and the start codon A in SEQ ID NO: 7. This means the 33rd position upstream from A. In addition, “upstream position 34 of the translation start point of the acpP gene” is the sequence of the start codon of the acpP gene when one base is inserted between the base at position 177 and the start codon A in SEQ ID NO: 7. It means the 35th position upstream from A.

In the acpP-fabF operon of an arbitrary bacterium, which base is the base “position 34 to the upstream of the translation start point of the acpP gene” can be determined by, for example, the upstream sequence of the bacterial acpP gene and SEQ ID NO: 7 This can be determined by performing alignment with the upstream sequence of the acpP gene. The alignment can be performed using, for example, known gene analysis software. Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et) al., Journal of molecular biology, 198 (2), 327-37. 1987).

Further, the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein. Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. The region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

In addition, gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

Also, gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Moreover, it is preferable that the reading frames of the sequences before and after the insertion site do not match. The other sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein. The host is transformed with the recombinant DNA containing, and the homologous recombination is caused between the deletion type gene and the wild type gene on the chromosome, thereby replacing the wild type gene on the chromosome with the deletion type gene. Can be achieved. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called “Red-driven integration” (Datsenko, K. A, and Wanner, B. L. Proc .Natl. Acad. Sci. U S A. 97: 6640-6645 (2000)), Red-driven integration method and λ phage-derived excision system (Cho, E. H., Gumport, R. I., Gardner, J F. J. Bacteriol. 184: 5200-5203 (2002)), a method using linear DNA such as a method (see WO2005 / 010175), a method using a plasmid containing a temperature-sensitive replication origin, There are a method using a plasmid capable of conjugation transfer and a method using a suicide vector which does not have an origin of replication and functions in a host (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

Further, the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like.

When the protein functions as a complex composed of a plurality of subunits, all of the plurality of subunits may be modified or only a part may be modified as long as the activity of the protein decreases as a result. . That is, for example, all of a plurality of genes encoding these subunits may be destroyed, or only a part of them may be destroyed. In addition, when a plurality of isozymes are present in a protein, as long as the activity of the protein is reduced as a result, all the activities of the plurality of isozymes may be reduced, or only a part of the activities may be reduced. That is, for example, all of a plurality of genes encoding these isozymes may be destroyed, or only a part of them may be destroyed.

The decrease in the activity of the protein can be confirmed by measuring the activity of the protein.

The decrease in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased, or confirming that the amount of protein expressed from the gene has decreased.

It can be confirmed that the amount of transcription of the gene has been reduced by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold spring spring Laboratory Laboratory, Cold spring Harbor (USA), 2001)). The amount of mRNA is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 55% or less, 50% or less, 30% or less, 20% or less, 10% or less as compared to the unmodified strain. It may be reduced to 5% or less, or 0%. However, the amount of mRNA transcribed from the acpP gene is not reduced to 0% compared to the unmodified strain. For example, when the expression of the acpP gene decreases, the amount of mRNA transcribed from the gene is 1% or more, 5% or more, 10% or more, 15% or more, 17% or more, compared to the unmodified strain, 20% or more, 30% or more, or 50% or more may remain. When the expression of the acpP gene decreases, the amount of mRNA transcribed from the gene is specifically 1% to 90%, 5% to 80%, 10% to It may be reduced to 70%, 15% to 60%, or 17% to 55%.

Confirmation that the amount of the protein has decreased can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Spring Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 55% or less, 50% or less, 30% or less, 20% or less, 10% or less compared to the unmodified strain It may be reduced to 5% or less, or 0%. However, the amount of AcpP protein is not reduced to 0% compared to the unmodified strain. For example, when the expression of the acpP gene decreases, the amount of AcpP protein is 1% or more, 5% or more, 10% or more, 15% or more, 17% or more, 20% or more, or compared to the unmodified strain, or 30% or more, or 50% or more may remain. When the expression of the acpP gene decreases, the amount of AcpP protein is specifically 1% to 90%, 5% to 80%, 10% to 70%, 15%, for example, as compared to the unmodified strain. It may be reduced to ˜60%, or 17% to 55%.

It can be confirmed that the gene has been destroyed by determining part or all of the nucleotide sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.

In addition to the weakening of the acpP-fabF operon, the above-described technique for reducing the activity of the protein generates a compound other than the target L-amino acid by branching from the biosynthetic pathway of any protein, for example, the target L-amino acid. It can be used to reduce the activity of an enzyme that catalyzes the reaction, or to reduce the expression of any gene, for example, a gene encoding any of these proteins.

<1-4> Technique for Increasing Protein Activity A technique for increasing protein activity is described below.

"" Protein activity increases "means that the activity per cell of the protein is increased relative to unmodified strains such as wild strains and parental strains. Note that “increasing protein activity” is also referred to as “enhancing protein activity”. “Protein activity increases” specifically means that the number of molecules per cell of the protein is increased and / or the function per molecule of the protein compared to an unmodified strain. Is increasing. That is, “activity” in the case of “increasing protein activity” means not only the catalytic activity of the protein, but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. “Protein activity increases” means not only to increase the activity of the protein in a strain that originally has the activity of the target protein, but also to the activity of the protein in a strain that does not originally have the activity of the target protein. Including granting. Further, as long as the activity of the protein increases as a result, the activity of the target protein inherent in the host may be reduced or eliminated, and the activity of a suitable target protein may be imparted.

The activity of the protein is not particularly limited as long as it is increased compared to the non-modified strain. For example, the protein activity is increased 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain. Good. In addition, when the non-modified strain does not have the activity of the target protein, it is sufficient that the protein is generated by introducing a gene encoding the protein. For example, the protein has an enzymatic activity. It may be produced to the extent that it can be measured.

Modification that increases the activity of the protein is achieved, for example, by increasing the expression of the gene encoding the protein. Note that “increasing gene expression” is also referred to as “enhanced gene expression”. The expression of the gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain. In addition, “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.

An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.

Increase in gene copy number can be achieved by introducing the gene into the host chromosome. Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller I, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having many copies on a chromosome include repetitive DNA sequences (inverted DNA) and inverted repeats present at both ends of a transposon. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for production of the target substance. Homologous recombination is, for example, the Red-driven integration method (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97: 6640-6645 (2000) ), A method using a linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, a method using a suicide vector that does not have a replication origin and functions in a host, or a phage It can be performed by the transduction method used. The gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1).

Confirmation of the introduction of the target gene on the chromosome was performed using Southern hybridization using a probe having a sequence complementary to all or part of the gene, or a primer prepared based on the sequence of the gene. It can be confirmed by PCR.

An increase in the copy number of a gene can also be achieved by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene can be linked to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the copy number of the gene. it can. A DNA fragment containing a target gene can be obtained, for example, by PCR using a genomic DNA of a microorganism having the target gene as a template. As the vector, a vector capable of autonomous replication in a host cell can be used. The vector is preferably a multicopy vector. In order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene. Moreover, the vector may be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. As vectors capable of autonomous replication in bacteria of the Enterobacteriaceae family such as Escherichia coli, specifically, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), wide host range Vector RSF1010 is mentioned.

In the case of introducing a gene, the gene may be retained in the bacterium of the present invention so that it can be expressed. Specifically, the gene may be introduced so as to be expressed under the control of a promoter sequence that functions in the bacterium of the present invention. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a native promoter of a gene to be introduced or a promoter of another gene. As the promoter, for example, a stronger promoter as described later may be used. Further, for example, a terminator for terminating transcription can be arranged downstream of the gene. The terminator is not particularly limited as long as it functions in the bacterium of the present invention. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator of another gene. Specific examples of the terminator include T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator. Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8, Genetic Engineering, Kyoritsu Shuppan, 1987”, and these can be used.

In addition, when two or more genes are introduced, each gene may be retained in the bacterium of the present invention so that it can be expressed. For example, all the genes may be held on a single expression vector, or all may be held on a chromosome. Moreover, each gene may be separately hold | maintained on several expression vector, and may be separately hold | maintained on the single or several expression vector and chromosome. Further, an operon may be constructed by introducing two or more genes. “When two or more genes are introduced” means, for example, when a gene encoding each of two or more enzymes is introduced, each of two or more subunits constituting a single enzyme is encoded. And a combination thereof.

The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The introduced gene may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, a primer designed based on the base sequence of the gene, and using a genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. The introduced gene may be totally synthesized based on the base sequence of the same gene (Gene, 60 (1), 115-127 (1987)).

When the protein functions as a complex composed of a plurality of subunits, all of the plurality of subunits may be modified or only a part may be modified as long as the activity of the protein increases as a result. . That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of a plurality of genes encoding those subunits may be enhanced, or only a part of the expression may be enhanced. Also good. Usually, it is preferable to enhance the expression of all of a plurality of genes encoding these subunits. In addition, each subunit constituting the complex may be derived from one organism or two or more different organisms as long as the complex has the function of the target protein. That is, for example, genes derived from the same organism encoding a plurality of subunits may be introduced into the host, or genes derived from different organisms may be introduced into the host.

Moreover, the increase in gene expression can be achieved by improving the transcription efficiency of the gene. Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter. By “stronger promoter” is meant a promoter that improves transcription of the gene over the native wild-type promoter. Examples of stronger promoters include the known high expression promoters T7 promoter, trp promoter, lac promoter, thr promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Can be mentioned. As a more powerful promoter, a highly active promoter of a conventional promoter may be obtained by using various reporter genes. For example, the activity of the promoter can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00/18935). Examples of the highly active promoter include various tac-like promoters (Katashkina JI et al. Russian Patent application 2006134574) and pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).

Moreover, the increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. By “a stronger SD sequence” is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence. As a stronger SD sequence, for example, RBS of gene 10 derived from phage T7 can be mentioned (Olins P. O. et al, Gene, 1988, 73, 227-235). Furthermore, substitution of several nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion, contributes to mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can be improved by modifying them.

In the present invention, promoters, SD sequences, and sites that affect gene expression such as a spacer region between the RBS and the start codon are also collectively referred to as “expression control regions”. The expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).

Improvement of gene translation efficiency can also be achieved, for example, by codon modification. In Escherichia coli, etc., there is a clear codon bias among the 61 amino acid codons found in the population of mRNA molecules, and the abundance of a tRNA seems to be directly proportional to the frequency of use of the corresponding codon. (Kane, JF, Curr. Opin. Biotechnol., 6 (5), 494-500 (1995)). That is, if a large amount of mRNA containing an excessive rare codon is present, translation problems may occur. Recent studies suggest that, inter alia, clusters of AGG / AGA, CUA, AUA, CGA, or CCC codons can reduce both the amount and quality of the synthesized protein. Such a problem can occur particularly during the expression of heterologous genes. Therefore, when performing heterologous expression of a gene, the translation efficiency of the gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. Codon substitution can be performed, for example, by a site-specific mutagenesis method in which a target mutation is introduced into a target site of DNA. As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, rlErlich, H. A. Eds., Stockton press (1989); Carter, P., ethMeth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth In Enzymol., 154, 367 (1987)). Alternatively, gene fragments in which codons have been replaced may be fully synthesized. The frequency of codon usage in various organisms can be found in the “Codon Usage Database” (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)) Is disclosed.

Also, the increase in gene expression can be achieved by amplifying a regulator that increases gene expression or by deleting or weakening a regulator that decreases gene expression.

The techniques for increasing gene expression as described above may be used alone or in any combination.

Further, the modification that increases the activity of the protein can be achieved, for example, by enhancing the specific activity of the protein. Specific activity enhancement also includes the reduction and elimination of feedback inhibition. Proteins with enhanced specific activity can be obtained by searching for various organisms, for example. Alternatively, a highly active protein may be obtained by introducing a mutation into a conventional protein. The introduced mutation may be, for example, a substitution, deletion, insertion or addition of one or several amino acids at one or several positions of the protein. Mutation can be introduced by, for example, the site-specific mutation method as described above. Moreover, you may introduce | transduce a variation | mutation by a mutation process, for example. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like. Alternatively, DNA may be directly treated with hydroxylamine in vitro to induce random mutations. The enhancement of specific activity may be used alone or in any combination with the above-described method for enhancing gene expression.

The method of transformation is not particularly limited, and a conventionally known method can be used. For example, as reported for Escherichia coli K-12, recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E .., 1997. Gene 1: 153-167) can be used. Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA. Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A. 1978. Nature 274: 398-400; Hinnen, A., Hicks, J. B. and Fink, G. R. 1978. Proc. Natl.Acad. Sci. USA 75: 1929-1933) can also be applied. Alternatively, an electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used.

The increase in protein activity can be confirmed by measuring the activity of the protein.

The increase in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has increased. An increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased, or by confirming that the amount of protein expressed from the gene has increased.

It can be confirmed that the transcription amount of the gene has increased by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. ), 2001). The amount of mRNA may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain.

Confirmation of the increase in the amount of protein can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain.

The above-described technique for increasing the activity of a protein can be used for enhancing the activity of an arbitrary protein, such as an L-amino acid biosynthetic enzyme, or for enhancing the expression of an arbitrary gene, such as a gene encoding the arbitrary protein.

<2> Method for Producing L-Amino Acid of the Present Invention The method of the present invention comprises culturing the bacterium of the present invention in a medium to produce and accumulate L-amino acid in the medium or in the microbial cells, and the medium. Alternatively, it is a method for producing an L-amino acid, which comprises collecting the L-amino acid from cells. In the present invention, one L-amino acid may be produced, or two or more L-amino acids may be produced.

The medium used is not particularly limited as long as the bacterium of the present invention can grow and L-amino acids are produced. As the medium, for example, a normal medium used for culturing microorganisms such as bacteria can be used. The medium may contain a component selected from a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and other various organic and inorganic components as necessary. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of bacteria used and the type of L-amino acid to be produced.

The carbon source is not particularly limited as long as it can be assimilated by the bacterium of the present invention to produce an L-amino acid. Specifically, as the carbon source, for example, glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolyzate, saccharides such as biomass hydrolyzate, acetic acid, fumaric acid, citric acid, Examples thereof include organic acids such as succinic acid and malic acid, alcohols such as glycerol, crude glycerol and ethanol, and fatty acids. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

The concentration of the carbon source in the medium is not particularly limited as long as the bacterium of the present invention can grow and L-amino acid is produced. The concentration of the carbon source in the medium is preferably as high as possible as long as the production of L-amino acid is not inhibited. The initial concentration of the carbon source in the medium may be, for example, usually 5 to 30% (W / V), preferably 10 to 20% (W / V). Moreover, you may add a carbon source additionally according to consumption of the carbon source accompanying progress of fermentation.

Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphoric acid source, one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

Other various organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

Moreover, when using an auxotrophic mutant strain that requires an amino acid or the like for growth, it is preferable to supplement nutrients required for the medium. For example, L-lysine producing bacteria often have an enhanced L-lysine biosynthetic pathway and weakened L-lysine resolution. Therefore, when culturing such L-lysine-producing bacteria, for example, one or more amino acids selected from L-threonine, L-homoserine, L-isoleucine, and L-methionine are supplemented to the medium. Is preferred.

In order to suppress foaming during culture, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.

Culture conditions are not particularly limited as long as the bacterium of the present invention can grow and L-amino acids are produced. The culture can be performed, for example, under normal conditions used for culture of microorganisms such as bacteria. The culture conditions may be appropriately set according to various conditions such as the type of bacteria used and the type of L-amino acid to be produced.

Cultivation can be performed using a liquid medium. When culturing, the culture medium of the bacterium of the present invention cultured in a solid medium such as an agar medium may be directly inoculated into a liquid medium, or the bacterium of the present invention seeded in a liquid medium is used as a liquid for main culture. The medium may be inoculated. That is, the culture may be performed separately for seed culture and main culture. The amount of the bacterium of the present invention contained in the medium at the start of culture is not particularly limited. For example, a seed culture solution having an OD660 of 4 to 8 may be added at 0.1 to 30% by mass, preferably 1 to 10% by mass with respect to the medium for main culture at the start of culture.

Culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. In addition, when culture is performed separately for seed culture and main culture, the culture conditions for seed culture and main culture may or may not be the same. For example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.

The culture can be performed aerobically, for example. For example, the culture can be performed by aeration culture or shaking culture. The oxygen concentration may be controlled to, for example, 5 to 50%, preferably about 10% of the saturated oxygen concentration. The pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary. The pH of the medium is adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. can do. The culture temperature may be, for example, 20 to 45 ° C, preferably 25 ° C to 37 ° C. The culture period may be, for example, 1 hour or more, 4 hours or more, 10 hours or more, or 15 hours or more, and may be 168 hours or less, 120 hours or less, 90 hours, or 72 hours or less. Specifically, the culture period may be, for example, 10 hours to 120 hours. The culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the bacterium of the present invention is lost. By culturing the bacterium of the present invention under such conditions, L-amino acids accumulate in the cells and / or in the medium.

In addition, when producing L-glutamic acid, it is also possible to carry out the culture while precipitating L-glutamic acid in the medium using a liquid medium adjusted to conditions under which L-glutamic acid is precipitated. The conditions under which L-glutamic acid precipitates are, for example, pH 5.0 to 3.0, preferably pH 4.9 to 3.5, more preferably pH 4.9 to 4.0, and particularly preferably around pH 4.7. (European Patent Application Publication No. 1078989). In addition, culture | cultivation may be performed at the said pH in the whole period, and may be performed at the said pH only for a part of period. The “partial period” may be, for example, a period of 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the entire culture period.

Further, when a basic amino acid such as L-lysine is produced, a method of fermenting basic amino acid using bicarbonate ion and / or carbonate ion as a main counter ion of basic amino acid may be used. (Unexamined-Japanese-Patent No. 2002-65287, US2002-0025564A, EP1813677A). According to these methods, basic amino acids can be produced while reducing the amount of sulfate ions and / or chloride ions that have been conventionally used as counter ions for basic amino acids.

The formation of L-amino acid can be confirmed by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used in appropriate combination.

The produced L-amino acid can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, and a crystallization method. These methods can be used in appropriate combination. In the case where L-amino acid accumulates in the microbial cells, for example, the microbial cells are crushed with ultrasonic waves, and the microbial cells are removed by centrifugation from the supernatant obtained by ion exchange resin method or the like. Amino acids can be recovered. The recovered L-amino acid may be a free form, a salt thereof, or a mixture thereof. Examples of the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt. For example, L-lysine may be free L-lysine, L-lysine sulfate, L-lysine hydrochloride, L-lysine carbonate, or a mixture thereof. Further, for example, L-glutamic acid may be free L-glutamic acid, sodium L-glutamate (MSG), ammonium L-glutamate, or a mixture thereof.

If L-amino acid is precipitated in the medium, it can be recovered by centrifugation or filtration. The L-amino acid precipitated in the medium may be isolated together after crystallization of the L-amino acid dissolved in the medium.

The recovered L-amino acid may contain components other than the L-amino acid, such as bacterial cells, medium components, water, and bacterial metabolic byproducts. The purity of the collected L-amino acid is, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 80% (w / w) or higher, 85% (JP1214636B, USP5,431,933, USP4,956,471, USP4,777,051, USP4,946,654, USP5,840,358, USP6,238,714 , US2005 / 0025878).

In one embodiment of the method of the present invention, Escherichia coli having L-lysine producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or in the microbial cells, and the medium or microbial cells A method for producing L-lysine, comprising collecting L-lysine, wherein the expression control sequence of the gene for the acpP-fabF operon is altered in Escherichia coli, whereby the expression of the gene is weakened It may be a method characterized by the above. Further, according to one aspect of the method of the present invention, Escherichia coli having L-lysine producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or in the microbial cells, and the medium or A method for producing L-lysine, comprising collecting L-lysine from bacterial cells, wherein cytosine at position −34 upstream of the translation start point of the acpP gene is substituted with another base in Escherichia coli. The method characterized by this may be used. Further, according to one aspect of the method of the present invention, Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or in the bacterial cells, and the medium or A method for producing L-lysine, comprising collecting L-lysine from a microbial cell, wherein cytosine at position −34 upstream of the translation start point of the acpP gene is substituted with adenine in the Escherichia coli. It may be a featured method. About these aspects of the method of this invention, the description regarding the bacteria of this invention mentioned above and the method of this invention can apply mutatis mutandis.

Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1: Construction of L-lysine-producing bacterium with reduced expression of acpP and fabF genes (1)
As an L-lysine producing bacterium, E. coli WC196ΔcadAΔldc strain (FERM BP-11027; WO2010 / 061890) (hereinafter also referred to as WC196LC strain) was used. A method called “Red-driven integration” (Proc. Natl. Acad. Sci. USA, 2000, vol. 1) was first developed by Datsenko and Wanner upstream of the acpP-fabF operon comprising the acpP and fabF genes of the same strain. 97, No. 12, p6640-6645). According to this method, the sequence corresponding to the target gene was designed on the 5 ′ side of the synthetic oligonucleotide, and the sequence corresponding to the antibiotic resistance gene was obtained using the synthetic oligonucleotide designed on the 3 ′ side. Mutation-introduced strains can be constructed in one step using PCR products. The procedure is shown below.

PCR was performed using the chromosomal DNA of Escherichia coli MG1655 strain (ATCC 47076) as a template and the synthetic oligonucleotides shown in SEQ ID NOs: 1 and 2 as primers. Primer of SEQ ID NO: 1, the 5 'end of the primer the sequence corresponding to the peripheral BglII site of the plasmid ^{pMW118 (λattL-Km r -λattR)} (WO2006 / 093322), corresponding to a portion of the upstream sequence of acpP gene sequence At the 3 'end of the primer. Primer of SEQ ID NO: 2, the 5 'end of the primer the sequence corresponding to the peripheral BglII site of the plasmid ^{pMW118 (λattL-Km r -λattR)} (WO2006 / 093322), corresponding to a portion of the downstream sequence of fabF gene sequence At the 3 'end of the primer. The resulting DNA fragment was ligated with the restriction enzymes BglII treated with vector pMW118 (λattL-Km ^r -λattR) and In-Fusion HD Cloning Kit (TAKARA BIO). E. coli JM109 was transformed with the In-Fusion reaction solution. Transformants were selected on L-agar medium containing 50 mg / L kanamycin. A plasmid was extracted from the transformant, and it was confirmed that the target fragment was inserted. This plasmid was designated ^{pMW118 (λattL-Km r -λattR)} -acpP-fabF.

pMW118 and (λattL-Km ^r -λattR) the -acpP-fabF template, the synthetic oligonucleotide shown in SEQ ID NO: 3 and 4 as primers, QuikChange Site-Directed Mutagenesis Kit plasmid introduced point mutations used (Agilent Technologies) Built. This mutation is a substitution of cytosine 34 bases upstream of the translation start point of the acpP gene with adenine. This plasmid was designated ^{pMW118 (λattL-Km r -λattR)} -acpP * -fabF.

and pMW118 the ^{(λattL-Km r -λattR) -acpP} * -fabF a template, using synthetic oligonucleotides shown in SEQ ID NO: 5 and 6 as primers, were PCR. Using the obtained DNA fragment, WC196LCacpP ^* strain was constructed from E. coli WC196LC strain using λ-red method described in US Patent Application Publication No. 2006/0160191 and WO2005 / 010175. In the WC196LCacpP ^* strain, cytosine 34 bases upstream of the translation start point of the acpP gene is replaced with adenine. Acquisition of kanamycin-resistant recombinants by the λ-red method was performed by plating on L-agar medium containing 50 mg / L kanamycin at 37 ° C. and selecting kanamycin-resistant recombinants.

Plasmid pCABD2 (US Pat. No. 6,040,160) was used to transform WC196LCacpP ^* strain, transformants were selected on L-agar medium containing 20 mg / L streptomycin, and WC196LCacpP ^* / pCABD2 strain was selected. Obtained. pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes.

Example 2: L-lysine production culture (1)
The prepared WC196LCacpP ^* / pCABD2 strain was used for L-lysine production culture. This strain was cultured at 37 ° C. in an L-agar medium containing 20 mg / L of streptomycin until the OD600 reached about 0.6, and then the same amount of 40% glycerol solution as the culture solution was added and stirred. Thereafter, an appropriate amount was dispensed and stored at −80 ° C. to prepare a glycerol stock.

A glycerol stock of WC196LCacpP ^* / pCABD2 strain was evenly spread on an L-agar medium containing 20 mg / L of streptomycin and cultured at 37 ° C. for 24 hours. The WC196LC / pCABD2 strain, which was a control strain in which pCABD2 was introduced into the WC196LC strain, was also cultured in the same manner on an L-agar medium containing 20 mg / L of streptomycin. The grown cells were suspended in 3.0 mL of an L-lysine production medium (MS-Glc medium) shown in Table 1, and the resulting suspension was diluted with the same medium so that the OD600 was 15. The obtained diluted suspension (1.0 mL) was inoculated into a 500 mL Sakaguchi flask in which 19 mL of L-lysine production medium containing 20 mg / L of streptomycin was infused, and was reconstituted using a reciprocating shake culture apparatus. Culturing was performed at 0 ° C. At 48 hours after the start of culture, the amount of remaining glucose and the amount of L-lysine produced were quantified.

Table 2 shows the residual glucose concentration and L-lysine accumulation concentration after 48 hours of culture. Compared with the control strain WC196LC / pCABD2, the WC196LCacpP ^* / pCABD2 strain in which the expression of the acpP and fabF genes was reduced significantly improved the L-lysine yield.

Example 3: Confirmation of expression level of acpP gene by RT-PCR (1)
The WC196LCacpP ^* / pCABD2 strain and WC196LC / pCABD2 strain prepared in Example 1 were each cultured under the conditions described in Example 2, and the culture solution was sampled at 17 hours of culture. RNA was extracted from the culture using RNAprotect Bacteria Reagent (Qiagen) and RNeasy Mini Kit (Qiagen). Using the obtained RNA as a template, reverse transcription PCR was performed using PrimeScript RT reagent Kit (Takara Bio). Using the obtained cDNA as a template, the synthetic oligonucleotides shown in SEQ ID NOs: 13 and 14 and the synthetic oligonucleotides shown in SEQ ID NOs: 15 and 16 as primers, and quantitative PCR using Power SYBR Green PCR Master Mix (Applied Biosystems) went. The oligonucleotides shown in SEQ ID NOs: 13 and 14 correspond to the base sequence of the acpP gene. The oligonucleotides shown in SEQ ID NOs: 15 and 16 correspond to sequences within the ORF of rrsA (16s rRNA). The mRNA amount of the acpP gene was calculated using rrsA (16s rRNA) as an internal standard.

Table 3 shows the amount of mRNA of the acpP gene at 17 hours of culture. The data are shown as relative values with the mRNA level of the acpP gene of the pCABD2 / WC196LC strain as 1. Compared with the control strain pCABD2 / WC196LC, the pCABD2 / WC196LC acpP ^* strain significantly decreased the amount of mRNA of the acpP gene.

Example 4: Construction of L-lysine-producing bacterium with reduced expression of acpP and fabF genes (2)
The WC196LC strain is used as an L-lysine producing bacterium. The upstream region of the acpP-fabF operon consisting of the acpP and fabF genes of the same strain is replaced with the P _tac84 promoter (Russian Patent Application Publication No. 2006/134574) or the lac promoter by the “Red-driven integration” method. The region to be replaced may be part or all of -200 to -1 upstream of the transcription start site of the acpP-fabF operon. For example, the region of -100 to -1, upstream -50 to -1, upstream -30 to -1, or upstream -30 to -10 upstream of the transcription start site of the acpP-fabF operon is replaced.

Hereinafter, a strain in which the region upstream of the acpP-fabF operon of the WC196LC strain is replaced with a P _tac84 promoter is referred to as a WC196LC P _tac84 acpP strain, and a strain in which the lac promoter is replaced is referred to as a WC196LC P _lac acpP strain.

WC196LC P _tac84 acpP and WC196LC P _lac acpP were transformed with plasmid pCABD2, and transformants were selected on L-agar medium containing 20 mg / L of streptomycin, and WC196LC P _tac84 acpP / pCABD2 And obtain WC196LC P _lac acpP / pCABD2 strain.

Example 5: L-lysine production culture (2)
Using the WC196LC P _tac84 acpP / pCABD2 strain and WC196LC P _lac acpP / pCABD2 strain prepared in Example 4 and the WC196LC / pCABD2 strain as a control strain, L-lysine production culture is performed according to the method described in Example 2.

Example 6: Confirmation of expression level of acpP gene by RT-PCR (2)
The WC196LC P _tac84 acpP / pCABD2 strain, the WC196LC P _lac acpP / pCABD2 strain, and the WC196LC / pCABD2 strain prepared in Example 4 were cultured under the conditions described in Example 2, respectively, and the method described in Example 3 was used. Calculate the amount of mRNA of the acpP gene.

Example 7: Construction of L-threonine-producing bacterium with reduced expression of acpP and fabF genes E. coli TDH-6 strain (Japanese Patent Laid-Open No. 2001-346578) is used as the L-threonine-producing bacterium. The TDH-6 strain can be obtained by removing the plasmid pVIC40 from E. coli TDH-6 / pVIC40 (VKPM B-3996) (Japanese Patent Laid-Open No. 2001-346578). Using the method described in Example 1, cytosine 34 bases upstream of the translation start point of the acpP gene of TDH-6 strain is replaced with adenine. Alternatively, using the method described in Example 4, the upstream region of the acpP-fabF operon of the TDH-6 strain is replaced with the P _tac84 promoter or the lac promoter. The region to be replaced may be part or all of -200 to -1 upstream of the transcription start site of the acpP-fabF operon. For example, the region of -100 to -1, upstream -50 to -1, upstream -30 to -1, or upstream -30 to -10 upstream of the transcription start site of the acpP-fabF operon is replaced.

Hereinafter, the TDH-6acpP ^* strain was substituted with cytosine 34 bases upstream of the translation start point of the adhP gene of the TDH-6 strain, and the upstream region of the acpP-fabF operon of the TDH-6 strain was used as the P _tac84 promoter. The substituted strain is called TDH-6 P _tac84 acpP strain, and the strain substituted with the lac promoter is called TDH-6 P _lac acpP strain.

Plasmid pVIC40 (US Pat. No. 5,705,371) was used to transform TDH-6acpP ^* strain, TDH-6 P _tac84 acpP strain, TDH-6 P _lac acpP strain, and TDH-6acpP ^* / pVIC40 strain, TDH-6 P _{A tac84} acpP / pVIC40 strain and a TDH-6 P _lac acpP / pVIC40 strain are obtained.

Example 8: L-threonine production culture TDH-6acpP ^* / pVIC40 strain, TDH-6 P _tac84 acpP / pVIC40 strain, TDH-6 P _lac acpP / pVIC40 strain prepared in Example 7, and TDH-6 as a control strain / pVIC40 strain is used for L-threonine production culture according to the method described in US Pat. No. 7,915,018.

Example 9: Confirmation of expression level of acpP gene by RT-PCR (3)
Using the TDH-6acpP ^* / pVIC40 strain, the TDH- _6P tac84 acpP / pVIC40 strain, the TDH-6P _lac acpP / pVIC40 strain prepared in Example 7 and the TDH-6 / pVIC40 strain as a control strain, L-threonine production culture is performed according to the method described in No. 7,915,018, and the mRNA amount of the acpP gene is calculated using the culture solution according to the method described in Example 3.

According to the present invention, the ability of bacteria to produce L-amino acids can be improved, and L-amino acids can be produced efficiently.

<Explanation of Sequence Listing>
SEQ ID NOs: 1 to 6: Primer SEQ ID NO: 7: base sequence of acpP-fabF operon of E. coli MG1655 and its upstream sequence SEQ ID NO: 8: amino acid sequence of AcpP protein of E. coli MG1655 SEQ ID NO: 9: of E. coli MG1655 The amino acid sequence of FabF protein SEQ ID NO: 10: the nucleotide sequence of the acpP-fabF operon of Pantoea ananatis AJ13355 and its upstream sequence SEQ ID NO: 11: the amino acid sequence of the AcpP protein of Pantoea ananatis AJ13355 SEQ ID NOs: 13 to 16: Primers

Claims (14)

1. Bacteria belonging to the family Enterobacteriaceae having L-amino acid-producing ability are cultured in a medium to produce and accumulate L-amino acid in the medium or in the bacterial body, and L-amino acid is produced from the medium or the bacterial body. Collecting an L-amino acid comprising:
   A method, characterized in that the bacterium has been modified such that the acpP-fabF operon is attenuated.
2. The method according to claim 1, wherein the weakening of the acpP-fabF operon is a decrease in the activity of a protein encoded by a gene of the acpP-fabF operon.
3. The method according to claim 1 or 2, wherein the operon is weakened by weakening the expression of the gene of the acpP-fabF operon.
4. 4. The method according to claim 3, wherein expression of the gene is weakened by modifying an expression control sequence of the gene of the acpP-fabF operon.
5. The method according to any one of claims 2 to 4, wherein the gene of the acpP-fabF operon is an acpP gene and / or a fabF gene.
6. The method according to claim 5, wherein the genes of the acpP-fabF operon are an acpP gene and a fabF gene.
7. 7. The expression of the acpP-fabF operon gene is attenuated by replacing cytosine at position −34 upstream of the translation start point of the acpP gene with another base. the method of.
8. The method according to claim 7, wherein the expression of the acpP-fabF operon gene is weakened by replacing cytosine at position −34 upstream of the translation start point of the acpP gene with adenine.
9. The method according to any one of claims 1 to 8, wherein the bacterium belongs to the genus Escherichia, Pantoea, or Enterobacter.
10. The method according to claim 9, wherein the bacterium is Escherichia coli.
11. The method according to any one of claims 1 to 10, wherein the L-amino acid is L-lysine.
12. Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or cells of the Escherichia coli, and L-lysine is collected from the medium or cells. A process for producing L-lysine comprising
    A method wherein the expression of the gene is weakened by modifying the expression control sequence of the gene of the acpP-fabF operon in the Escherichia coli.
13. Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or cells of the Escherichia coli, and L-lysine is collected from the medium or cells. A process for producing L-lysine comprising
    A method wherein cytosine at position −34 upstream of the translation start point of the acpP gene is substituted with another base in the Escherichia coli.
14. Escherichia coli having L-lysine-producing ability is cultured in a medium, and L-lysine is produced and accumulated in the medium or cells of the Escherichia coli, and L-lysine is collected from the medium or cells. A process for producing L-lysine comprising
    A method wherein cytosine at position −34 upstream of the translation start point of the acpP gene is substituted with adenine in the Escherichia coli.